Isolation and characterization of DNA repetitions carrying the chromosomal beta-lactamase gene of Escherichia coli K-12.

Summary A ColE1 hybrid plasmid, pNU1, carrying the amp operon coding for chromsomal -lactamase was isolated from the Clarke and Carbon collection and physically mapped. The physical location of ampC within this plasmid was further deduced by in vitro cloning.By reciprocal recombination between pNU1 and chromosome of two unstable -lactamase hyperproducing E. coli K-12 mutants a large plasmid from each mutant was obtained. The respective plasmid was physically mapped and found to contain five and two repeated DNA segments. The repetitions within each plasmid were equal in size, 9,800 bp and 11,900 bp respectively and were organized in tandem. The end points of the repeats were different in the two plasmids but shared a DNA segment carrying the ampC gene. The chromosomal DNA of the -lactamase hyperproducing E. coli mutants were found to contain an amplified DNA segment equal in size to the repeated unit found in the respective plasmid. The data shows that up to 10 identical repeats organized in tandem can be generated by a normal mutation frequency in E. coli.     

Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or) [ and ]. Inactivation or deletion of or results in SegA^– plasmids. Better than random segregation requires an active segB region. The segB region contains two ors (or and or). Inactivation of either of the orfs does not lead to an increase in cell death, but or^– plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.     

Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions

Summary The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.     

Role of homology and pathway specificity for recombination between plasmids and bacteriophage lambda

Summary To determine the minimum amount of homology required for efficient recombination in Escherichia coli, we measured recombination frequencies between bacteriophage and pBR322 derivatives containing DNA fragments of various sizes by assaying for phages that could transduce the bla and ori genes of pBR322. Efficient recombination required about 40 bp of homology; increases in homology above 40 bp resulted in proportionate increases in recombination, while decreases below 40 bp resulted in precipitous decreases in recombination. The recA ⁺ gene stimulated recombination over the entire range of homologies tested. Restriction enzyme digests of several recombinant DNA molecules indicated that they contained the complete plasmid DNA inserted in the genome as expected for a reciprocal crossover. Analysis of recombination frequencies in different recombination-deficient mutant strains indicated that the formation of -plasmid cointegrates by homologous recombination proceeded predominantly by the RecBC pathway and very inefficiently, if at all, by the RecE and RecF pathways.     

The genetic defect in the various types of human β-galactosidase deficiency

Summary Gene localization studies revealed the presence of two structural -galactosidase (GAL) loci on the human chromosomes 3 and 22 (de Wit et al., 1979). To determine the function of these genes, proliferating hybrid cell lines were isolated following fusion of fibroblasts from two different patients with a GAL deficiency and Chinese hamster cells. The hybrids were analyzed electrophoretically and immunologically.Fibroblasts from a patient with an adult type of GAL deficiency associated with a neuraminidase deficiency were used for the first fusion. No evidence for a structural GAL mutation was found in these hybrids. The absence of a structural GAL mutation is consistent with a primary defect in neuraminidase in this adult patient.Fibroblasts from a patient with the infantile type 1 G_M1-gangliosidosis were used for the second fusion. It is concluded that the human determinants present in the isolated hybrid lines occur in heteropolymeric man-Chinese hamster molecules. The heteropolymeric isoenzyme in (+3–22) hybrids is very labile and is sensitive to neuraminidase treatment. Therefore it is concluded that the infantile type 1 patient is mutated in the structural GAL gene on chromosome 3. Because this patient has a primary defect in G_M1-GAL, the GAL gene on chromosome 3 is apparently a G _M1-GAL gene. Interaction of the two GAL loci results in an additional band of GAL activity on electrophoresis. This suggests that the gene on chromosome 22 is also a structural G _M1-GAL gene.     

A 7.1 kbp beta-myosin heavy chain promoter,efficient for green fluorescent protein expression,probably induces lethality when overexpressing a mutated transforming growth factor-beta type II receptor in transgenic mice

The roles of transforming growth factor-beta (TGF) in heart or skeletal muscle development and physiology are still the subject of controversies. Our aim was to block, in transgenic mice, the TGF signalling pathway by a dominant negative mutant of the TGF type II receptor fused to the enhanced green fluorescent protein (TRII-KR-EGFP) under the control of a 7.1 kbp mouse beta-myosin heavy chain (MHC) promoter to investigate the roles of TGF in the heart and slow skeletal muscles. First, we generated two transgenic lines overexpressing EGFP under the control of the 7.1 kbp MHC promoter. In embryos, EGFP was detectable as early as 7.5 days post coitum. In embryos, newborns and adults, EGFP was expressed mainly in the cardiac ventricles and in slow skeletal muscles. EGFP expression was intense in the bladder but weak in the intestines. In contrast to the endogenous MHC promoter, the activity of the 7.1 kbp MHC promoter in the transgene was not repressed after birth and remained high in adult transgenic mice. We obtained two founders with the transgene comprising the TRII-KR-EGFP sequence under the control of the 7.1 kbp MHC promoter. These founders were generated at a very low frequency and expressed barely detectable levels of TRII-KR-EGFP mRNA. Our failure to obtain transgenic lines overexpressing the dominant negative receptor suggests that the blocking of the TGF signalling pathway in the heart and slow skeletal muscles could be embryonically lethal. To conclude, the 7.1 kbp MHC promoter directs high levels of transgene expression in the cardiac ventricles and in slow skeletal muscles of the mouse. Analysis of the consequences of the blocking of the TGF signalling pathway in the heart will require the use of tissue specific means of conditional gene invalidation.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Primary structure of the marmoset (Saguinus fuscicollis) hemoglobin. I. Use of tryptic maleylated peptides in the solubilization and sequence elucidation of the α- and β-chains

The primary structure of adult marmoset hemoglobin has been determined. The - and -chains of HbA were separated on a CM23 column in 8 M urea using a sodium phosphate gradient. Tryptic digests of the - and -chains were fractionated on a Dowex 50W-X2 column using a pH and pyridine acetate gradient. Large peptide fragments were obtained by the cyanogen bromide cleavage of the - and -chains, as well as by tryptic digestion of the maleylated - and -chains. The sequence was derived from the amino acid compositions and sequences of the individual tryptic peptide, automated sequence determination of intact - and -chains, as well as automated sequence determination of cyanogen bromide fragments and tryptic maleylated peptides derived from the - and -chains. The complete structure of marmoset adult hemoglobin is closely homologous to that of other primate hemoglobins. The sequence of the marmoset -chain differs from the -chain of human HbA at positions 8, 19, 23, 68, and 116. The -chain from marmoset HbA differs from the -chain of human HbA at positions 5, 13, 21, 50, 87, and 125.This work was supported in part by funds from a Physicians' Medical Education and Research Foundation Grant of the University of Tennessee Memorial Research Hospital and by NIH General Research Support Grant FR-5541 to the institution.     

β-D-Glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1→3),(1→4)β-D-glucan synthase

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (14)-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four `U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called `hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species,Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a `class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (14)-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (14)-glycan synthases in plants. For example, the mixed-linkage (13)(14)-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other -linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage -glucan synthases not only provide special insight into the mechanisms of (14)-glycan synthesis but may also uncover the genes that encode the synthases themselves.     

Particle bombardment-mediated transient expression of a Brazil nut methionine-rich albumin in bean (Phaseolus vulgaris L.)

Bean (Phaseolus vulgaris L.) mature embryos were transformed using biolistic methods with a plasmid containing 2S albumin and -glucuronidase structural sequences, both under the control of the 35S CaMV promoter. We have shown that chimaeric tissues could be obtained and that both structural sequences were expressed to similar levels.     

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Organization and structure of Volvox beta-tubulin genes

Summary Genomic clones encoding two Volvox -tubulin genes have been isolated and shown to represent the only two -tubulin genes in the genome. Restriction fragment length polymorphism analysis was used to demonstrate that the two genes are genetically linked. One of these genes was sequenced and the mRNA start site(s) determined by primer extension. A comparison of its sequence to those of the two -tubulin genes of Chlamydomonas revealed: (1) a high degree of conservation of the coding region, with the predicted amino acid sequence differing only in the C-terminal residue; (2) extensive sequence conservation in the 5 untranslated leader region and a 16 bp (putative regulatory) sequence in the promoter region; (3) the same number and location of introns, with a short region of homology in intron 1, but little significant homology in introns 2 and 3.     

Meiotic recombination in an Irish family with beta-thalassaemia

Using the technique of allele-specific priming of the polymerase chain reaction (PCR), the C-T substitution in codon 39 was identified as the cause of -thalassaemia in an Irish family. Analysis of the restriction fragment length polymorphisms (RFLPs) in the -globin gene cluster established linkage of the -thalassaemia mutation to a particular -haplotype but indicated that a recombinational event had occurred in the paternal chromosome in the younger of two affected children. Non-paternity was excluded by DNA fingerprinting analysis with hypervariable minisatellite probes. This is the fourth case of recombination in the -globin gene cluster to be reported. The event has occurred 5 of the polymorphic RsaI site at position-550 bp upstream of the -globin gene mRNA Cap site, within the 9.1-kb region that has been shown to be a hot spot for recombination in the -globin gene cluster.     

Further evidence for abnormal protein kinase C regulation of macromolecule secretion in fibroblasts from cystic fibrosis patients

In comparison to skin fibroblasts from normal subjects, those from patients with cystic fibrosis (CF): (1) bound [20-³H] phorbol 12,13-dibutyrate (PDBu) with a higher affinity (K_d=25.8 vs 12.8 nM respectively) but expressed a similar number of total phorbol ester binding sites (about 2.5 pmol PDBu bound/mg of protein); (2) exhibited a faster and higher response to 4-phorbol 12-myristate 13-acetate (PMA) for the stimulation of [³⁵S]-labelled glycoconjutate release, but were equally sensitive to the synergistic effect of A23187 on this process; and (3) secreted glycoconjugates with similar [³⁵S]-sulfate and [¹⁴C]-leucine to [¹⁴C]-glucosamine labelling ratios. Taken together, these results provide further evidence for abnormal protein kinase C (PKC) regulation of macromolecule secretion in CF disease.Abbreviations BSA Bovine serum albumin - DBcAMP Dibutyryl cyclic AMP - DMEM Dulbecco's modified Eagle's medium - DMSO Dimethylsulfoxide - LDH Lactate dehydrogenase - PBS Phosphate-buffered saline - PDBu 4-phorbol 12,13-dibutyrate - 4-PDD 4-phorbol 12,13-didecanoate - PMA 4-phorbol 12-myristate 13-acetate - TCA Trichloroacetic acid     

Evolution of a protein superfamily: Relationships between vertebrate lens crystallins and microorganism dormancy proteins

Summary A search of sequence databases shows that spherulin 3a, an encystment-specific protein ofPhysarum polycephalum, is probably structurally related to the - and -crystallins, vertebrate ocular lens proteins, and to Protein S, a sporulation-specific protein ofMyxococcus xanthus. The - and -crystallins have two similar domains thought to have arisen by two successive gene duplication and fusion events. Molecular modeling confirms that spherulin 3a has all the characteristics required to adopt the tertiary structure of a single -crystallin domain. The structure of spherulin 3a thus illustrates an earlier stage in the evolution of this protein superfamily. The relationship of - and -crystallins to spherulin 3a and Protein S suggests that the lens proteins were derived from an ancestor with a role in stressresponse, perhaps a response to osmotic stress.     

A putative β-glucanase pseudogene behind the potato GBSS gene

We identified an open reading frame (ORF) which is located closely behind the gene encoding granulebound starch synthase (GBSS) of potato (Solanum tuberosum L.). The ORF ends with a perfect 43 bp direct repeat, which carries the stop triplet precisely at the beginning of the second repeat. The deduced protein shows homology with all known isoforms of plant -1,3-glucanases and -1,3-1,4-glucanases. Although the DNA sequence is unique in potato and tomato (Lycopersicon esculentum L.), no expression of the gene was found in these species. Taken together with the unusual codon usage and length of the predicted protein, this sequence could be a pseudogene.     

Interaction of PRK1 Receptor-like Kinase with a Putative elF2B β-Subunit in Tobacco

PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata. We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins. The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast -subunit of translation initiation factor 2B (eIF2B-), which was designated NeIF2B. eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells. Deletion mutants of NeIF2B were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2B, especially the region between residue 103 and 235, is important for the interaction. This protein association was confirmed by in vitro binding assay of the recombinant NeIF2B and PRK1 proteins. Despite high sequence homology between NeIF2B and its yeast counterpart, the NeIF2B cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-, when transferred into the mutant strain.     

High degree of homology of the deduced protein structures of the tetracycline-resistance determinants between Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5

The amino acid sequences of the tetracycline-resistance (Tc^r) determinants of Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5 have been deduced from their nucleotide sequences and compared. The deduced Tc^r proteins (TETs) of pNS1981 (458 amino acids) and pTP5 (459 amino acids) show a considerable homology (60% identical). If homologous amino acid replacement is taken into account, the homology becomes 80%. Both TET proteins are highly hydrophobic, as expected for a membrane-binding protein, and their polarities are calculated at 32–33%. The putative secondary structures of both TET proteins have been also shown to be significantly homologous, being abundant in -sheets. The predicted positions of -sheets show a nice coincidence between both TET proteins. -Helix has a tendency to be formed at nonhomologous regions of the primary structures between both TET proteins. However, the predicted positions of -helices coincide in a frequency greater than 50%. -Helix and random coil moderately occur at the hydrophilic regions in both TET proteins.     

Location by fluorescence microscopy of glycosidases and a xylanase in the anaerobic gut fungi Caecomyces communis,Neocallimastix frontalis,and Piromyces rhizinflata

-d-Glucosidase, -d-fucosidase -d-xylosidase, and -cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. -d-Galactosidase and -l-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi.